The present invention concerns a method for the detection of cytosine methylation in DNA samples.
The levels of observation that have been well studied due to method developments in recent years in molecular biology include the genes themselves, as well as transcription and translation of these genes into RNA and the proteins arising there from. During the course of development of an individual, when a gene is turned on and how the activation and inhibition of certain genes in certain cells and tissues are controlled can be correlated with the extent and nature of the methylation of the genes or of the genome. Pathogenic states are also expressed by a modified methylation pattern of individual genes or of the genome.
5-Methylcytosine is the most frequent covalently modified base in the DNA of eukaryotic cells. For example, it plays a role in the regulation of transcription, in genetic imprinting and in tumorigenesis. The identification of 5-methylcytosine as a component of genetic information is thus of considerable interest. 5-Methylcytosine positions, however, cannot be identified by sequencing, since 5-methylcytosine has the same base-pairing behavior as cytosine. In addition, in the case of a PCR amplification, the epigenetic information, which is borne by 5-methylcytosines, is completely lost.
A relatively new method that has since been applied most frequently for investigating DNA for 5-methylcytosine is based on the specific reaction of bisulfite with cytosine, which is converted to uracil, which corresponds in its base-pairing behavior to thymidine, after a subsequent alkaline hydrolysis. In contrast, 5-methylcytosine is not modified under these conditions. Thus the original DNA is converted, such that methylcytosine, which originally cannot be distinguished from cytosine by means of its hybridization behavior, now can be detected by “standard” molecular biological techniques as the single remaining cytosine, for example, by amplification and hybridization or sequencing. All of these techniques are based on base pairing, which now is fully utilized. The prior art, which concerns sensitivity, is defined by a method that incorporates the DNA to be investigated in an agarose matrix, through which diffusion and renaturation of the DNA is prevented (bisulfite reacts only on single-stranded DNA) and all precipitation and purification steps are replaced by rapid dialysis (Olek A., Oswald J., Walter J. A modified and improved method for bisulphate based cytosine methylation analysis. Nucleic Acids Res. 1996 Dec. 15; 24 (24): 5064-6). Individual cells can be investigated with this method, which illustrates the potential of the method. Of course, previously, only individual regions of up to approximately 3000 base pairs in length have been investigated; a global investigation of cells for thousands of possible methylation analyses is not possible. Of course, this method also cannot reliably analyze very small fragments of small sample quantities. These are lost despite the protection from diffusion through the matrix.
A review of the other known possibilities for detecting 5-methylcytosines can be derived from the following review article: Rein T, DePamphilis M L, Zorbas H. Identifying 5-methylcytosine and related modifications in DNA genomes. Nucleic Acids Res. 1998 May 15; 26 (10): 2255-64.
The bisulfite technique has previously been applied only in research, with a few exceptions (e.g., Zeschnigk M, Lich C, Buiting K, Dörfler W, Horsthemke B. A single-tube PCR test for the diagnosis of Angelman and Prade-Willi syndrome based on allelic methylation differences at the SNRPN locus. Eur J Hum Genet. 1997 March-April; 5 (2): 94-8). However, short, specific pieces of a known gene are always amplified after a bisulfite treatment and either completely sequenced (Olek A, Walter J. The pre-implantation ontogeny of the H19 methylation imprint. Nat Genet. 1997 November.; 17(3): 275-6) or individual cytosine positions (Gonzalgo M L, Jones P A. Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Res. 1997 Jun. 15;25 (12):2529-31; WO 95/00669) or an enzyme cleavage (Xiong Z, Laird P W. COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res. 1997 Jun. 15;25(12): 2532-4) are detected by a “primer extension reaction”. Also, detection by means of hybridizing has been described (Olek et al., WO 99/28498).
Urea improves the efficiency of the bisulfite treatment prior to the sequencing of 5-methylcytosine in genomic DNA (Paulin R, Grigg G W, Davey M W, Piper A A. Urea improves efficiency of bisulphate-mediated sequencing of 5′-methylcytosine in genomic DNA Nucleic Acids Res. 1998 November 1;26(21):5009-10).
Other publications, which are concerned with the application of the bisulfite technique for the detection of methylation in individual genes are: Grigg G, Clark S. Sequencing 5-methylcytosine residues in genomic DNA. Bioassays. 1994 June; 16(6):431-6, 431; Zeschnigk M, Schmitz B, Dittrich B, Buiting K, Horsthemke B, Dörfler W. Imprinted segments in the human genome: different DNA methylation patterns in the Prader-Willi/Angelman syndrome region as determined by the genomic sequencing method. Hum Mol Genet. 1997 March; 6(3):387-95; Feil R, Charlton J, Bird A P, Walter J, Reik W. Methylation analysis on individual chromosomes: improved protocol for bisulphate genomic sequencing. Nucleic Acids Res. 1994 February 25;22(4):695-6; Martin V, Ribieras S, Song-Wang X, Rio M C, Dante R. Genomic sequencing indicates a correlation between DNA hypomethylation in the 5′ region of the pS2 gene and in its expression in human breast cancer cell lines. Gene. May 19, 1995; 157 (1-2): 261-4; WO 97/46705, WO 95/15373 and W097/45560.
Another known method is so-called methylation-sensitive PCR (Herman J G, Graff J R, Myohanen S, Nelkin B D, Baylin S B. (1996), Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA. Sep. 3; 93 (18): 9821-6). For this method, primers are used, which hybridize either only to a sequence that is formed by the bisulfite treatment of a DNA that is not methylated at the respective position, or, vice versa, primers, which only bind to a nucleic acid which has formed by the bisulfite treatment of a DNA that is methylated at the respected position. With these primers, amplified products can then be produced, whose detection in turn supplies hints of the presence of a methylated or unmethylated position in the sample, to which the primers bind.
A more recent method is also the detection of cytosine methylation by means of a TaqMan PCR, which has become known as “methyl-light” (WO 00/70090). It is possible with this method to detect the methylation status of individual positions or a few positions directly in the course of the PCR, so that a subsequent analysis of the products is spared.
A review of the prior art in oligomer array production can be taken from a special publication of Nature Genetics that appeared in January 1999 (Nature Genetics Supplement, Volume 21, January 1999), the literature cited therein and U.S. Pat. No. 5,994,065 on methods for the production of solid carriers for target molecules such as oligonucleotides with reduced nonspecific background signal.
Probes with many fluorescent labels have been used for the scanning of an immobilized DNA array. Particularly suitable for fluorescent labels is the simple introduction of Cy3 and Cy5 dyes at the 5′-OH of the respective probe. The fluorescence of the hybridized probes is detected, for example, by means of a confocal microscope. The dyes Cy3 and Cy5, in addition to many others, are commercially available.
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF) is a very powerful development for the analysis of biomolecules (Karras M, Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem. 1998 Oct. 15;60 (20): 2299-301). An analyte is embedded in a light-absorbing matrix. The matrix is evaporated by means of a short laser pulse and the analyte molecule is transported unfragmented into the gas phase. The ionization of the analyte is achieved by collisions with matrix molecules. An applied voltage accelerates the ions in a field-free flight tube. The ions are accelerated to a varying extent based on their different masses. Smaller ions reach the detector sooner than larger ions.
MALDI-TOF spectroscopy is excellently suitable for the analysis of peptides and proteins. The analysis of nucleic acids is somewhat more difficult (Gut, I. G. and Beck, S. (1995), DNA and Matrix Assisted Laser Desorption Ionization Mass Spectrometry. Molecular Biology: Current Innovations and Future Trends 1: 147-157.) For nucleic acids, the sensitivity is approximately 100 times poorer than for peptides and decreases overproportionally with increasing fragment size. For nucleic acids, which have a backbone with multiple negative charges, the ionization process through the matrix is essentially less efficient. In MALDI-TOF spectroscopy, the selection of the matrix plays a very important role. For the desorption of peptides, several very powerful matrices have been found, which produce a very fine crystallization. Several high-performing matrices have been found in the meantime for DNA, but the difference in sensitivity has not been reduced in this way. The difference in sensitivity can be reduced by modifying the DNA chemically in such a way that it is similar to a peptide. Phosphorothioate nucleic acids, in which the usual phosphates of the backbone are substituted by thiophosphates, can be converted into a charge-neutral DNA by simple alkylation chemistry (Gut, I. G. and Beck, S. (1995), A procedure for selective DNA alkylation and detection by mass spectrometry. Nucleic Acids Res. 23: 1367-1373). The coupling of a “charge tag” to this modified DNA results in an increase in sensitivity by the same amount that is found for peptides. Another advantage of “charge tagging” is the increased stability of the analysis against impurities, which greatly interfere with the detection of unmodified substrates.
Genomic DNA is obtained by standard methods from DNA of cells, tissue or other test samples.
This standard methodology is found in references such as Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 1989.
Accordingly, up until now there have been many methods for methylation analysis in the prior art. The present invention, however, will solve the problem that current methods are unable to solve, i.e., to amplify in a targeted manner a DNA to be investigated that is found in a body fluid or serum, when other, sequence-homologous DNA segments of different origin are also present.
The DNA to be investigated as well as the otherwise present nucleic acids, which are named background DNA below, are usually amplified equally, since the primers used are not able to distinguish between the DNA to be investigated and background DNA. One possibility for distinguishing these DNAs, however, is by the different methylation pattern. A current method is methylation-sensitive PCR, abbreviated MSP (Herman J G, Graff J R, Myohanen S, Nelkin B D, Baylin S B. (1996), Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA. September 3; 93 (18): 9821-6). This method is comprised of several steps. First, a bisulfite treatment is conducted according to the prior art, which in turn leads to the circumstance that all cytosine bases are converted to uracil while the methylated cytosine bases (5-methylcytosine) remain unchanged. In the next step, one now uses primers, which are completely complementary to a methylated DNA converted with bisulfite, but not to a corresponding DNA which was present originally in the unmethylated state. When the PCR is conducted with such a primer, this leads to the fact that the originally methylated DNA is amplified exclusively. It is also possible to use a primer, which, in contrast, amplifies only unmethylated DNA. In this way, if the DNA to be analyzed as well as background DNA are present, the DNA fragments to be investigated will be produced selectively and exclusively, as long as they differ from the background DNA relative to their methylation status in a CpG position. Prior art is now to infer from the detection of such a DNA molecule to be investigated either the methylation state or the presence of a DNA to be investigated, which in turn permits, in principle, a diagnosis, for example, of a tumor disorder in the patient, since it is known, for example, that the serum DNA concentration is in part drastically increased in tumor patients. Only the DNA originating from tumors will be detected in addition to the background DNA. In principle, the analysis of DNA is comparable in other body fluids.
The method that is described here and which is to be considered as the closest prior art, however, has several disadvantages. For example, it is not possible to conclude the quantity present in serum from the detectability of an amplified fragment of the DNA to be investigated. Even minimal quantities of such DNA are successful for obtaining a positive result, which is an advantage, on the one hand, but can also act in a very unfavorable manner, if one wishes to evaluate, for example, the effect of a tumor resection on serum DNA. The greatest disadvantage, however, is that many methylation positions are present, in which the DNA to be investigated and the background DNA differ only in degree. It is obvious that the existing MSP method can only be conducted if one knows that the backbround DNA differs definitively and up to 100% from the DNA to be investigated in the CpG position of interest, if one does not want to risk false positive results. In contrast, it is typical in a tumor tissue that a specific position is present in the methylated state, e.g., in 95% of the tumor cells, in which the otherwise present background DNA, however, is present in the methylated state only to a maximum of 5%, so that it is not possible with the MSP method to produce informative results, since a quantification of the template DNA by means of PCR in principle is not possible or can be accomplished only with increased expenditure. Also, this invention is based on the knowledge that often methylation patterns are present in a DNA fragment, which are typical for a specific type of cell, for example, a tumor cell.
Also, prior art includes a process developed by Epigenomics, which amplifies equally the DNA to be investigated and background DNA after bisulfite treatment and then examines the former CpG positions contained in the fragment by hybridization techniques, and alternatively by means of mini-sequencing or another current method. This has the advantage, that a quantitative pattern is obtained relative to the investigated methylated positions, i.e., the determination of the methylation degree of a multiple number of positions is successfully obtained, which, e.g., makes possible a very accurate classification in the case of solid tumors. The disadvantage of this method, however, is that it cannot supply accurate information in those cases in which the background DNA greatly predominates, since this information is amplified accurately along with the DNA to be investigated and both are analyzed in the mixture. This problem does not exist in the analysis of solid tumors, in which the material to be investigated can be selected in a targeted manner, but the analysis, for example, of serum DNA is made difficult.